Unfractionated heparin (UFH) inhibits EHV-1-induced platelet activation, as measured by P-selectin expression with flow cytometry. (A) Platelets were gated (R1) in a forward versus side scatter plot (left panel) and the percentage of P-selectin-positive cells in the gated region was measured (marker or M1 region) using histogram plots of P-selectin fluorescence (right panel). In the absence of UFH, thrombin (0.15 U/mL) and both RacL11 and Ab4 strains of EHV-1 at 1 PFU/cell activated platelets. Activation was evident as lengthening and narrowing of the platelet event cloud, with some microvesiculation (arrows) and >90% platelets expressing P-selectin. (B) In the presence of 2 U/mL UFH, inhibition of platelet activation is seen in forward versus side scatter (left panel) and histogram plots of P-selectin expression (right panel) in gated platelets (R1) exposed to thrombin and both viruses. No changes are seen in the PBS control with or without UFH. Residual small events (<101 forward scatter units) in the virus-exposed samples in the presence of UFH likely represent aggregated virus particles. Images in (A,B) are representative of one horse. Note that EHV-1-induced activation in this horse was not completely abolished in the presence of heparin, possibly due to mild “preactivation” of platelets (4% P-selectin expression in PBS control). (C) Quantification of the median percentage of P-selectin-positive platelets (columns), with superimposed individual data points, in response to thrombin (light gray columns) or both strains of EHV-1 (RacL11, dark gray columns; Ab4, black columns) in the absence or presence of 2 U/mL UFH (n = 6–7). *Numbers shown are P values (Wilcoxon matched pairs sign rank). (D) A heparin dose titration curve showed consistent inhibition of thrombin (light gray columns) and EHV-1-induced platelet activation at 0.05 U/mL (RacL11, dark gray columns; Ab4, black columns; n = 3–10). Columns represent medians with superimposed individual data points.

The phosphodiesterase inhibitors, IBMX and cilostazol, reduce EHV-1-induced platelet activation, as measured by P-selectin expression with flow cytometry. P-selectin expression in response to thrombin (0.15 U/mL, light gray columns) or RacL11 (dark gray columns) and Ab4 (black columns) strains of EHV-1 at 1 PFU/cell was quantified as the percentage of gated platelets in the absence or presence of increasing concentrations of (A) IBMX (5–50 μM) (n = 3–4) or (B) cilostazol (cilo, 1–50 μM) (n = 3–9). Both drugs inhibited activation induced by thrombin and both virus strains, to varying degrees. The DMSO vehicle control mildly suppressed the activation responses. No activation was seen with the PBS control in the absence or presence of either drug (only highest dose of drug shown). Bars represent medians with superimposed individual data points. (C) Forward versus side scatter plots show the inhibitory effects of IBMX as well as the platelet fragmentation or vesiculation that occurred in the presence of the DMSO vehicle and virus (arrows), but not thrombin. A similar vesiculation effect was seen with cilostazol (not shown). Increased small events (<101 forward scatter units) in the virus alone-exposed samples represent a combination of microvesicles or platelet fragments and aggregates of virus particles. The number of replicates was insufficient to perform statistical analysis.